Automated process line

ABSTRACT

Fully automated modular analytical systems with integrated instrumentation for analysis of biopolymer samples, such as nucleic acids, proteins, peptides and carbohydrates, are provided. Analytical methods of detection and analysis, such as mass spectrometry, radiolabeling, mass tags, chemical tags and fluorescence chemiluminescence, are integrated with robotic technology and automated chemical reaction systems to provide a high-throughput, accurate Automated Process Line (APL).

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 09/285,481to Hubert Köster, Ping Yip, Jhobe Steadman, Dirk Reuter and RichardMacDonald, filed Apr. 2, 1999, entitled “AUTOMATED PROCESS LINE” isclaimed. U.S. application Ser. No. 09/285,481 is incorporated byreference in its entirety.

BACKGROUND OF THE INVENTION

In recent years, developments in the field of life sciences haveproceeded at a breathtaking rate. Ground breaking scientific discoveriesand advances in such fields as genomics (sequencing and characterizationof genetic information and analysis of the relationship between geneactivity and cell function) and proteomics (systematic analysis ofprotein expression in tissues, cells, and biological systems) promise toreshape the fields of medicine, agriculture, and environmental science.The success of these efforts depends, in part, on the development ofsophisticated laboratory tools that will automate and expedite thetesting and analysis of biological samples.

Current methods of testing typically employ multiple instruments forpreparing and analyzing samples and involve multiple manual handlingsteps and transfers. Such procedures are labor-intensive,time-consuming, and costly and they are susceptible to human error,sample contamination, and loss. After samples have been prepared, theycan be subjected to testing procedures that produce data for analysis.Conventional testing procedures often must be performed by an individuallaboratory technician, one sample at a time. Laboratory technicians aretypically individuals who are most likely trained to operate only asingle instrument. Automation will reduce the number of personnel andtraining necessary to carry out the research. Reliable and accurateautomated process and analysis tools are necessary for the benefits ofrecent scientific discoveries to be fully achieved.

Genomic research is increasing the availability of genomic markers thatcan be used for the identification of all organisms, including humans.These markers (all genetic loci including SNPs, microsatellites andother noncoding genomic regions) provide a way to not only identifypopulations but also allow stratification of populations according totheir response to drug treatment, resistance to environmental agents,and other factors. Importantly, the identification of the large numberof genomic markers has become the driving force behind the developmentof new automated technologies.

At the forefront of the efforts to develop better analytical tools areefforts to expedite the analysis of complex biochemical structures. Forexample, robotic devices have been employed to assist in samplepreparation and handling.

Such automated sample preparation systems could find application is theareas of: identification and validation of disease-causing genes or drugtargets; defining mutations and polymorphisms associated with specificdiseases; monitoring gene expression and comparing disease states, cellcycles or other changes; genetic profiling of patients forresponsiveness to genomics-based therapies; and genetic profiling ofsubjects in drug clinical studies to link response with genotype.

The utility of genomic markers to identify and stratify populations isdepending on the industry's ability to measure great numbers(100-100,000) of markers in large populations. This approach isextremely limited in terms of time and research costs. Automation ofthese systems provides advantages such as increasing throughput andaccuracy, but miniaturization also is an important consideration interms of research costs. Accordingly, there is a need to automateprocesses in which very small volumes are handled, and retain theaccuracy of the results to permit their use in high throughput screeningprotocols and diagnostics.

Therefore it is an object herein to provide automated systems andmethods for high-throughput analysis of biological samples, particularlysamples of very small volume, for screening, diagnosis and otherprocedures. Other objects will become apparent from the followingdisclosure.

SUMMARY OF THE INVENTION

Provided herein is a fully automated modular analytical system thatintegrates sample preparation, instrumentation, and analysis ofbiopolymer samples. The samples include, but are not limited to, allbiopolymers, e.g., nucleic acids, proteins, peptides, carbohydrates, PNA(peptide nucleic acids), biopolymer (nucleic acid/peptide) analogs, andlibraries of combinatorial molecules. The system integrates analyticalmethods of detection and analysis including but are not limited to, massspectrometry, radiolabeling, mass tags, chemical tags, fluorescencechemiluminescence, with robotic technology and automated chemicalreaction systems to provide a high-throughput, accurate automatedprocess line (APL). The systems and methods provided herein areparticularly suited for handling very small volumes, on the order ofmilliliters, nanoliters and even smaller picoliter volumes.

In certain embodiments, the analytical system includes one portion thatis a contamination-controlled environment, such as a clean room orlaminar flow room, and includes a means, such as a transporter, formoving the samples from such environment into a second room or space forfurther processing. This dual space system permits performance ofprocedures that require clean room conditions to be automatedly linkedto procedures that do not require such conditions.

An integrated system for performing a process line comprising aplurality of processing stations, each of which performs a procedure ona biological sample contained in a reaction vessel; a robotic systemthat transports the reaction vessel from processing station toprocessing station; a control system that determines when the procedureat each processing station is complete and, in response, moves thereaction vessel to the next test station, and continuously processesreaction vessels one after another until the control system receives astop instruction; and a data analysis system that receives test resultsof the process line and automatically processes the test results to makea determination regarding the biological sample in the reaction vesselis provided.

The APL can run unattended continuously with a continuous samplethroughput and is capable of analyzing on the order of 10,000-50,000genotypes per day. The results are highly accurate and reproducible.

Also provided herein are methods for automated analysis of biopolymersusing the integrated APL system. In preferred embodiments, provided areautomated methods for preparing a biological sample for analysis;introducing the sample into an analytical instrument; recording sampledata; automatically processing and interpreting the data; and storingthe data in a bioinformatics database. In a particular embodiment,patient DNA samples are automatically analyzed to determine genotype.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of the components of the automated process line.

FIG. 2 shows a magnetic strip construction of the magnetic liftillustrated in FIG. 1.

FIG. 3 shows a point-magnet construction of the magnetic liftillustrated in FIG. 1.

FIG. 4 shows the robotic interface between the chip processor and themass spectrometer of the automated process line illustrated in FIG. 1.

FIG. 5 shows a comparison of a mass spectrum of a test sample withstored spectra from samples with known genotypes.

FIG. 6 is a flow diagram that illustrates the data analysis processingsteps performed by the automated process line of FIG. 1.

FIG. 7 shows an example of the user interface to the APL system.

FIG. 8 shows an example of the interface to a database of experimentalmass spectral data.

DETAILED DESCRIPTION AND PREFERRED EMBODIMENTS Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs. All patents, patent applicationsand publications referred to herein are, unless noted otherwise,incorporated by reference in their entirety. In the event a definitionin this section is not consistent with definitions elsewhere, thedefinition set forth in this section will control.

As used herein, a molecule refers to any molecule or compound that islinked to the bead. Typically such molecules are macromolecules orcomponents or precursors thereof, such as peptides, proteins, smallorganics, oligonucleotides or monomeric units of the peptides, organics,nucleic acids and other macromolecules. A monomeric unit refers to oneof the constituents from which the resulting compound is built. Thus,monomeric units include, nucleotides, amino acids, and pharmacophoresfrom which small organic molecules are synthesized.

As used herein, macromolecule refers to any molecule having a molecularweight from the hundreds up to the millions. Macromolecules includepeptides, proteins, nucleotides, nucleic acids, and other such moleculesthat are generally synthesized by biological organisms, but can beprepared synthetically or using recombinant molecular biology methods.

As used herein, a biological particle refers to a virus, such as a viralvector or viral capsid with or without packaged nucleic acid, phage,including a phage vector or phage capsid, with or without encapsulatednucleotide acid, a single cell, including eukaryotic and prokaryoticcells or fragments thereof, a liposome or micellar agent or otherpackaging particle, and other such biological materials. For purposesherein, biological particles include molecules that are not typicallyconsidered macromolecules because they are not generally synthesized,but are derived from cells and viruses.

As used herein, the term “nucleic acid” refers to single-stranded and/ordouble-stranded polynucleotides such as deoxyribonucleic acid (DNA), andribonucleic acid (RNA) as well as analogs or derivatives of either RNAor DNA. Also included in the term “nucleic acid” are analogs of nucleicacids such as peptide nucleic acid (PNA), phosphorothioate DNA, andother such analogs and derivatives.

As used herein, the term “biological sample” refers to any materialobtained from any living source (e.g., human, animal, plant, bacteria,fungi, protist, virus). For purposes herein, the biological sample willtypically contain a nucleic acid molecule. Examples of appropriatebiological samples include, but are not limited to: solid materials(e.g., tissue, cell pellets, biopsies) and biological fluids (e.g.,urine, blood, saliva, amniotic fluid, mouth wash, cerebral spinal fluidand other body fluids).

As used herein, the phrases “chain-elongating nucleotides” and“chain-terminating nucleotides” are used in accordance with their artrecognized meaning. For example, for DNA, chain-elongating nucleotidesinclude 2′deoxyribonucleotides (e.g., dATP, dCTP, dGTP and dTTP) andchain-terminating nucleotides include 2′,3′-dideoxyribonucleotides(e.g., ddATP, ddCTP, ddGTP, ddTTP). For RNA, chain-elongatingnucleotides include ribonucleotides (e.g., ATP, CTP, GTP and UTP) andchain-terminating nucleotides include 3′-deoxyribonucleotides (e.g.,3′dA, 3′dC, 3′dG and 3′dU). A complete set of chain elongatingnucleotides refers to dATP, dCTP, dGTP and dTTP. The term “nucleotide”is also well known in the art.

As used herein, nucleotides include nucleoside mono-, di-, andtriphosphates. Nucleotides also include modified nucleotides such asphosphorothioate nucleotides and deazapurine nucleotides. A complete setof chain-elongating nucleotides refers to four different nucleotidesthat can hybridize to each of the four different bases comprising theDNA template.

As used herein, “multiplexing” refers to the simultaneously detection ofmore than one analyte, such as more than one (mutated) loci on aparticular captured nucleic acid fragment (on one spot of an array).

As used herein, the term “biopolymer” is used to mean a biologicalmolecule composed of two or more monomeric subunits, or derivativesthereof, which are linked by a bond or a macromolecule. A biopolymer canbe, for example, a polynucleotide, a polypeptide, a carbohydrate, or alipid, or derivatives or combinations thereof, for example, a nucleicacid molecule containing a peptide nucleic acid portion or aglycoprotein, respectively. The methods and systems herein, thoughdescribed with reference to biopolymers, can be adapted for use withother synthetic schemes and assays, such as organic syntheses ofpharmaceuticals, or inorganics and any other reaction or assay performedon a solid support or in a well in nanoliter volumes.

As used herein, the term “nucleic acid” refers to single-stranded and/ordouble-stranded polynucleotides such as deoxyribonucleic acid (DNA), andribonucleic acid (RNA) as well as analogs or derivatives of either RNAor DNA. Also included in the term “nucleic acid” are analogs of nucleicacids such as peptide nucleic acid (PNA), phosphorothioate DNA, andother such analogs and derivatives.

As used herein, the term “polynucleotide” refers to an oligomer orpolymer containing at least two linked nucleotides or nucleotidederivatives, including a deoxyribonucleic acid (DNA), a ribonucleic acid(RNA), and a DNA or RNA derivative containing, for example, a nucleotideanalog or a “backbone” bond other than a phosphodiester bond, forexample, a phosphotriester bond, a phosphoramidate bond, aphosphorothioate bond, a thioester bond, or a peptide bond (peptidenucleic acid). The term “oligonucleotide” also is used hereinessentially synonymously with “polynucleotide,” although those in theart will recognize that oligonucleotides, for example, PCR primers,generally are less than about fifty to one hundred nucleotides inlength.

Nucleotide analogs contained in a polynucleotide can be, for example,mass modified nucleotides, which allows for mass differentiation ofpolynucleotides; nucleotides containing a detectable label such as afluorescent, radioactive, luminescent or chemiluminescent label, whichallows for detection of a polynucleotide; or nucleotides containing areactive group such as biotin or a thiol group, which facilitatesimmobilization of a polynucleotide to a solid support. A polynucleotidealso can contain one or more backbone bonds that are selectivelycleavable, for example, chemically, enzymatically or photolytically. Forexample, a polynucleotide can include one or more deoxyribonucleotides,followed by one or more ribonucleotides, which can be followed by one ormore deoxyribonucleotides, such a sequence being cleavable at theribonucleotide sequence by base hydrolysis. A polynucleotide also cancontain one or more bonds that are relatively resistant to cleavage, forexample, a chimeric oligonucleotide primer, which can includenucleotides linked by peptide nucleic acid bonds and at least onenucleotide at the 3′ end, which is linked by a phosphodiester bond, orthe like, and is capable of being extended by a polymerase. Peptidenucleic acid sequences can be prepared using well known methods (see,for example, Weiler et al., Nucleic acids Res. 25:2792-2799 (1997)).

A polynucleotide can be a portion of a larger nucleic acid molecule, forexample, a portion of a gene, which can contain a polymorphic region, ora portion of an extragenic region of a chromosome, for example, aportion of a region of nucleotide repeats such as a short tandem repeat(STR) locus, a variable number of tandem repeats (VNTR) locus, amicrosatellite locus or a minisatellite locus. A polynucleotide also canbe single stranded or double stranded, including, for example, a DNA-RNAhybrid, or can be triple stranded or four stranded. Where thepolynucleotide is double stranded DNA, it can be in an A, B, L or Zconfiguration, and a single polynucleotide can contain combinations ofsuch configurations.

As used herein, the term “polypeptide,” means at least two amino acids,or amino acid derivatives, including mass modified amino acids and aminoacid analogs, that are linked by a peptide bond, which can be a modifiedpeptide bond. A polypeptide can be translated from a polynucleotide,which can include at least a portion of a coding sequence, or a portionof a nucleotide sequence that is not naturally translated due, forexample, to it being located in a reading frame other than a codingframe, or it being an intron sequence, a 3′ or 5′ untranslated sequence,a regulatory sequence such as a promoter, or the like. A polypeptidealso can be chemically synthesized and can be modified by chemical orenzymatic methods following translation or chemical synthesis. The terms“polypeptide,”“peptide” and “protein” are used essentially synonymouslyherein, although the skilled artisan will recognize that peptidesgenerally contain fewer than about fifty to one hundred amino acidresidues, and that proteins often are obtained from a natural source andcan contain, for example, post-translational modifications. Apolypeptide can be post-translationally modified, such as byphosphorylation (phosphoproteins) and glycosylation (glycoproteins,proteoglycans), in a cell or in a reaction in vitro.

As used herein, the term “conjugated” refers stable attachment,preferably ionic or covalent attachment. Among preferred conjugationmeans are: streptavidin- or avidin- to biotin interaction; hydrophobicinteraction; magnetic interaction (e.g., using functionalized magneticbeads, such as DYNABEADS, which are streptavidin-coated magnetic beadssold by Dynal, Inc. Great Neck, N.Y. and Oslo Norway); polarinteractions, such as “wetting” associations between two polar surfacesor between oligo/polyethylene glycol; formation of a covalent bond, suchas an amide bond, disulfide bond, thioether bond, or via crosslinkingagents; and via an acid-labile or photocleavable linker.

As used herein equivalent, when referring to two sequences of nucleicacids means that the two sequences in question encode the same sequenceof amino acids or equivalent proteins. When “equivalent” is used inreferring to two proteins or peptides, it means that the two proteins orpeptides have substantially the same amino acid sequence with onlyconservative amino acid substitutions that do not substantially alterthe activity or function of the protein or peptide. When “equivalent”refers to a property, the property does not need to be present to thesame extent [e.g., two peptides can exhibit different rates of the sametype of enzymatic activity], but the activities are preferablysubstantially the same. “Complementary,” when referring to twonucleotide sequences, means that the two sequences of nucleotides arecapable of hybridizing, preferably with less than 25%, more preferablywith less than 15%, even more preferably with less than 5%, mostpreferably with no mismatches between opposed nucleotides. Preferablythe two molecules will hybridize under conditions of high stringency.

As used herein: stringency of hybridization in determining percentagemismatch are those conditions understood by those of skill in the artand typically are substantially equivalent to the following:

1) high stringency: 0.1×SSPE, 0.1% SDS, 65° C.

2) medium stringency: 0.2×SSPE, 0.1% SDS, 50° C.

3) low stringency: 1.0×SSPE, 0.1% SDS, 50° C.

It is understood that equivalent stringencies may be achieved usingalternative buffers, salts and temperatures.

As used herein, a primer when set forth in the claims refers to a primersuitable for mass spectrometric methods requiring immobilizing,hybridizing, strand displacement, sequencing mass spectrometry refers toa nucleic acid must be of low enough mass, typically about 70nucleotides or less than 70, and of sufficient size to be useful in themass spectrometric methods described herein that rely on massspectrometric detection. These methods include primers for detection andsequencing of nucleic acids, which require a sufficient numbernucleotides to from a stable duplex, typically about 6-30, preferablyabout 10-25, more preferably about 12-20. Thus, for purposes herein aprimer will be a sequence of nucleotides comprising about 6-70, morepreferably a 12-70, more preferably greater than about 14 to an upperlimit of 70, depending upon sequence and application of the primer. Theprimers herein, for example for mutational analyses, are selected to beupstream of loci useful for diagnosis such that when performing usingsequencing up to or through the site of interest, the resulting fragmentis of a mass that sufficient and not too large to be detected by massspectrometry. For mass spectrometric methods, mass tags or modifier arepreferably included at the 5′-end, and the primer is otherwiseunlabeled.

As used herein, “conditioning” of a nucleic acid refers to modificationof the phosphodiester backbone of the nucleic acid molecule (e.g.,cation exchange) for the purpose of eliminating peak broadening due to aheterogeneity in the cations bound per nucleotide unit. Contacting anucleic acid molecule with an alkylating agent such as akyliodide,iodoacetamide, β-iodoethanol, or 2,3-epoxy-1-propanol, the monothiophosphodiester bonds of a nucleic acid molecule can be transformed intoa phosphotriester bond. Likewise, phosphodiester bonds may betransformed to uncharged derivatives employing trialkylsilyl chlorides.Further conditioning involves incorporating nucleotides that reducesensitivity for depurination (fragmentation during MS) e.g., a purineanalog such as N7- or N9-deazapurine nucleotides, or RNA building blocksor using oligonucleotide triesters or incorporating phosphorothioatefunctions that are alkylated or employing oligonucleotide mimetics suchas peptide nucleic acid (PNA).

As used herein, the term “solid support” means a non-gaseous, non-liquidmaterial having a surface. Thus, a solid support can be a flat surfaceconstructed, for example, of glass, silicon, metal, plastic or acomposite; or can be in the form of a bead such as a silica gel, acontrolled pore glass, a magnetic or cellulose bead; or can be a pin,including an array of pins suitable for combinatorial synthesis oranalysis.

As used herein, substrate refers to an insoluble support onto which asample is deposited according to the materials described herein.Examples of appropriate substrates include beads (e.g., silica gel,controlled pore glass, magnetic, agarose gel and crosslinked dextrose(i.e. Sepharose and Sephadex, cellulose and other materials known bythose of skill in the art to serve as solid support matrices. Forexamples substrates may be formed from any or combinations of: silicagel, glass, magnet, polystyrene/1% divinylbenzene resins, such as Wangresins, which are Fmoc-aminoacid-4-(hydroxymethyl)phenoxy-methylcopoly(styrene-1% divinylbenzene(DVD)) resin, chlorotrityl (2-chlorotritylchloride copolystyrene-DVBresin) resin, Merrifield (chloromethylated copolystyrene-DVB) resinmetal, plastic, cellulose, cross-linked dextrans, such as those soldunder the trade name Sephadex (Pharmacia) and agarose gel, such as gelssold under the trade name Sepharose (Pharmacia), which is a hydrogenbonded polysaccharide-type agarose gel, and other such resins and solidphase supports known to those of skill in the art. The support matricesmay be in any shape or form, including, but not limited to: capillaries,flat supports such as glass fiber filters, glass surfaces, metalsurfaces (steel, gold, silver, aluminum, copper and silicon), plasticmaterials including multiwell plates or membranes (e.g., ofpolyethylene, polypropylene, polyamide, polyvinylidenedifluoride), pins(e.g., arrays of pins suitable for combinatorial synthesis or analysisor beads in pits of flat surfaces such as wafers (e.g., silicon wafers)with or without plates, and beads. The supports include any supportsused for retaining or conjugating macromolecules and biopolymers, andbiological particles.

As used herein, a selectively cleavable linker is a linker that iscleaved under selected conditions, such as a photocleavable linker, achemically cleavable linker and an enzymatically cleavable linker (i.e.,a restriction endonuclease site or a ribonucleotide/RNase digestion).The linker is interposed between the support and immobilized DNA.

As used herein, the term “liquid dispensing system” means a device thatcan transfer a predetermined amount of liquid to a target site. Theamount of liquid dispensed and the rate at which the liquid dispensingsystem dispenses the liquid to a target site, which can contain areaction mixture, can be adjusted manually or automatically, therebyallowing a predetermined volume of the liquid to be maintained at thetarget site.

As used herein, the term “liquid” is used broadly to mean a non-solid,non-gaseous material, which can be homogeneous or heterogeneous and cancontain one or more solid or gaseous materials dissolved or suspendedtherein. In general, a liquid is a component of a reaction mixture thatis susceptible to evaporation under the conditions of the reaction. Inparticular, the liquid can be a solvent, in which a reaction isperformed, for example water or glycerol/water or buffer or reactionmixture, where the reaction is performed in an aqueous solution. Theliquid can be any non-solid, non-gaseous solvent or other component of areaction mixture that is susceptible to evaporative loss, for example,acetonitrile, which can be a solvent for a nucleic acid synthesisreaction; formamide, which can be a liquid component of a nucleic acidhybridization reaction; piperidine, which is a liquid component of anucleic acid sequencing reaction; or any other non-aqueous solvent orother liquid component. A liquid can contain dissolved or suspendedcomponents, which can be useful, for example, for initiating,terminating or changing the conditions of a reaction, therebyfacilitating the performance of single tube reactions.

As used herein, the term “reaction mixture” refers to any solution inwhich a chemical, physical or biological change is effected. In general,a change to a molecule is effected, although changes to cells also arecontemplated. A reaction mixture can contain a solvent, which provides,in part, appropriate conditions for the change to be effected, and asubstrate, upon which the change is effected. A reaction mixture alsocan contain various reagents, including buffers, salts, and metalcofactors, and can contain reagents specific to a reaction, for example,but are not limited to, enzymes, nucleoside triphosphates and aminoacids. For convenience, reference is made herein generally to a“component” of a reaction, wherein the component can be a cell ormolecule present in a reaction mixture, including, for example, abiopolymer or a product thereof.

As used herein, the term “target site” refers to a specific locus on asolid support that can contain a liquid. A solid support contains one ormore target sites, which can be arranged randomly or in ordered array orother pattern. In particular, a target site restricts growth of a liquidto the “z” direction of an xyz coordinate. Thus, a target site can be,for example, a well or pit, a pin or bead, or a physical barrier that ispositioned on a surface of the solid support, or combinations thereofsuch as a beads on a chip, chips in wells, or the like. A target sitecan be physically placed onto the support, can be etched on a surface ofthe support, can be a “tower” that remains following etching around alocus, or can be defined by physico-chemical parameters such as relativehydrophilicity, hydrophobicity, or any other surface chemistry thatallows a liquid to grow primarily in the z direction. A solid supportcan have a single target site, or can contain a number of target sites,which can be the same or different, and where the solid support containsmore than one target site, the target sites can be arranged in anypattern, including, for example, an array, in which the location of eachtarget site is defined.

As used herein, the term “predetermined volume” is used to mean anydesired volume of a liquid. For example, where it is desirable toperform a reaction in a 5 microliter volume, 5 microliters is thepredetermined volume. Similarly, where it is desired to deposit 200nanoliters at a target site, 200 nanoliters is the predetermined volume.

As used herein, a small volume, typically refers to a volume on theorder of nanoliters, preferably less than 1 microliter and typically,less than 0.5 microliters and less. The term nanoliter volume refers toa volume of about 0.1 to about 1000 nanoliters, preferably about 1 to100 nanoliters.

As used herein, symbology refers to the code, such as a bar code, thatis engraved or imprinted on a surface. The symbology is any code knownor designed by the user.

As used herein, a bar codes refers any array of, preferably, opticallyreadable marks of any desired size and shape that are arranged in areference context or frame of, preferably, although not necessarily, oneor more columns and one or more rows. For purposes herein, the bar coderefers to any symbology, not necessary “bar” but may include dots,characters or any symbol or symbols.

As used herein, the disclosed systems and methods generally are usefulwhere the reaction volume is about 500 milliliters or less; are moreuseful where the reaction volume is about 5 milliliters or less; aremost useful where the reaction volume is in the “submilliliter” range,for example, about 500 microliters, or about 50 microliters or about 5microliters or less; and are particularly useful where the reactionvolume is a “submicroliter” reaction volume, which can be measured innanoliters, for example, about 500 nanoliters or less, or 50 nanolitersor less or 10 nanoliters or less, or can be measured in picoliters, forexample, about 500 picoliters or less or about 50 picoliters or less.For convenience of discussion, the term “submicroliter” is used hereinto refer to a reaction volume less than about one microliter, althoughit will be readily apparent to those in the art that the systems andmethods disclosed herein are applicable to subnanoliter reaction volumesas well.

As used herein, a room refers to a space, such as a room, chamber or ahood or other enclosure that is in some manner separated. In anembodiment herein, the APL system is designed to operate in two rooms,such that manipulations that require sterile conditions can be performedin one room or chamber. Manipulations that do not require suchconditions can be performed in a second room. Samples can then beautomatically transported between the first room and second room. Asdesired additional rooms, with conditions designed for a particular setof manipulations may be included in the system.

Automated Process Line

In the Automated Process Line (APL) constructed in accordance with thedisclosure herein, one or more robotic systems under computer controlare used to manipulate the sample of interest. The robot(s) arecommanded by controlling software and move the sample between the seriesof reaction and sample preparation stations that comprise the APL. Therobot includes a robotic arm that moves, for example, along a track oron a central pivot, and is typically outfitted with a “gripper” arm,allowing it to grip reaction vessels and transport them betweenstations. Such robotic systems are commercially available and arecommonly known to those of skill in the art. For example, a roboticsystem and accompanying software can be obtained from Robocon Labor-undIndustrieroboter Ges.m.b.H of Austria (“Robocon”). In a preferredembodiment, the APL includes a Robocon “Model CRS A 255” robot, equippedwith a “Digital Servo Gripper” mechanism, also available from Robocon.The robotic systems are designed such that they can be integrated withother computer-controlled instrumentation to perform consecutiveoperations to effect a multi-step process.

In the preferred embodiment, one robot moves along a central track in acontamination-controlled environment, such as a positive airflow orlaminar flow chamber, to perform a series of manipulations or reactionson a biological sample. Once these steps are completed, the sampleenters a second contamination-controlled environment, which serves as anantechamber into a non-sterile environment. The second environment canbe sealed off from the first contamination-controlled environment and/orthe non-sterile environment. For example, in a particular embodiment,the sample is transported from the contamination-controlled laminar flowchamber into a transport chamber, or taxicab. If desired, the taxicabcan provide a sterile environment.

Upon entry of the sample into the transport chamber, thecontamination-controlled environment is sealed off. The sample thenmoves along a pneumatically-driven or motor-driven stage in thetransport chamber, and the transport chamber then opens up into the,non-sterile environment, such as an open room. In the open room, asecond robot, also moving along a central track, takes control ofmanipulating the sample.

The sample to be analyzed is contained within a reaction vessel that isdesigned to integrate with all of the components of the APL and which isamenable to the conditions of the chemical or biological reactionsperformed. Preferred for high throughput analysis are reaction vesselsthat are capable of containing multiple samples, such as multiwellmicrotiter plates, preferably 96-well or 384-well plates or chips, suchas silicon microchips. The reaction vessels also can comprise flat chipswith reaction sites which are not wells, but physical locations thatcontain the reaction using a chemical barrier. In certain embodiments,the robot and/or gripper is adapted to hold a sample vessel. Forexample, pins may be added to the gripper in alignment with the wells ofa microtiter plate for transporting the sample.

In high-throughput applications, where multiple sample plates are to beanalyzed successively in an automated fashion, the samples can be heldin a sample storage system, or rack, where they are picked up by thesystem robot and processed. An example of such a sample storage system,for use with multi-well microtiter plates, is the Robocon “Plate Cube”system.

In steps where sample vessels are to be sealed, such as when subjectedto PCR amplification, or unsealed, such as for reagent addition orremoval, an automated lid application/removal and sealing system may beintegrated into the system. Examples of these include a lid parkingstation, such as is available from Robocon, and a plate sealer, such asthe “MJ Microseal”, available from MJ Research. A system turntable mightalso be employed to assist the system robot in orienting the samples fordelivery into each station of the APL. Such a turntable is available,for example, from Robocon. Additionally, a shaker is also included inthe APL system in embodiments where beads or other reagents are added tothe sample for immobilizing the sample, or where other manipulationsrequiring mechanical shaking are involved.

In preferred embodiments, the sample plate or vessel is coded with asymbology, such as a bar code, which can be read by a reader, to allowsample tracking. In the preferred embodiment, separate bar code readersare contained in the contamination-controlled and non-sterileenvironments. Bar code systems, including one and two dimensional barcodes, readable and readable/writable codes and systems therefor, arewidely available, such as from Datalogic S.p.A. of Italy (“Datalogic”),and are well known to those skilled in the art.

Sample handling and reagent additions are accomplished using automatedliquid handling systems. These include systems capable of automaticallydispensing liquids into the sample vessel, such as through a pipette,and can be adapted to any sample format, such as a multiwell microtiterplate. Such systems are commercially available, such as from Tecan AG ofSwitzerland (“Tecan”) or Beckman Coulter, Inc. In a preferredembodiment, Tecan “Genesis 200/8” (200 cm with including an 8-tip arm)liquid handling systems, as well as a Beckman Coulter “Multimek 96”automated pipettor are used for liquid handling. Other liquid dispensingsystems are described in allowed U.S. application Ser. Nos. 08/787,639,08/786,988, and published International PCT application No. WO 98/20166,which are incorporated herein by reference.

Also present in the system may be an apparatus for preparing a testsample for analysis, including, for example, reagent addition means, orother means for performing reactions or processes to prepare the samplefor analysis. In certain preferred embodiments, where mass spectralanalysis, specifically MALDI-TOF analysis, is to be performed using asample array, a matrix material (i.e., an organic acid) is added to thesample using an adapted piezoelectric pipetting dispensing system. Thedispensing system includes a hydrophobic tip, which is capable ofdispensing submolar, preferably nanomolar, samples. Such systems, aswell as methods for preparing and analyzing low volume analyte arrayelements, have been described in allowed U.S. patent application Ser.Nos. 08/787,639, 08/786,988, and published International PCT applicationNo. WO 98/20166, see, also Little et al., Anal. Chem. 1997, 69,4540-4546, the contents of which are incorporated by reference herein intheir entirety.

Alternatively, a system that dispenses liquid samples from the picoliterup to the nanoliter range is commercially available, such as the“Nano-Plotter” product from GeSiM GmbH of Germany (“GeSiM”). In otherembodiments, reactions such as radiolabeling or adding a mass tag to thesample may be performed by the sample preparation apparatus.

A sample may also be transferred to or placed in a particular sampleanalysis vessel for analysis. The particular type of sample analysisvessel used is determined by the analytical method to be employed. Forexample, in a preferred embodiment, where mass spectrometry (MALDI-TOF)is used for analysis of a sample, a typical sample vessel is a siliconmicrochip (<1 square inch) that includes one or more, 100, 200, 300,400, 500, up to 999 diagnostic sites, or even higher density, on asingle chip, preferably in the pattern of a 2-D array. The chip, ormultiple chips, can then be placed on a sample platform, designedspecifically to be inserted into the mass spectrometer.

In a preferred embodiment, the analytical system is a MALDI-TOF massspectrometer. A preferred mass spectrometer is manufactured byBruker-Franzen Analytik GmbH of Germany (“Bruker”) and uses a UV laser.In the spectrometer, a brief pulse of laser irradiation is absorbed bythe matrix, leading to spontaneous volatilization and ionization of thematrix and DNA fragments. The molecular weight of the gas-phase ions arethen determined by measurement of the time-of-flight of ions, which isproportional to their mass.

It should be understood that the nature of the sample to be analyzed andthe analysis to be performed, as well as the feasibility of automating areaction process, determine the components integrated into the APL, andthe system is not to be limited to the particular embodiments describedherein.

Module for Performing the Reaction in an Unsealed Environment

Systems for performing a reaction in an unsealed environment areprovided in copending U.S. application Ser. No. 09/266,409, filed Mar.10, 1999. These systems may be integrated into the APL provided herein.Briefly the systems and methods provide a means of maintaining a volumeof a liquid, for example, a reaction mixture, present in an unsealedenvironment and, therefore, susceptible to loss of volume byevaporation. The liquid generally is present on a surface of a solidsupport, at a target site, and the environment into which evaporationcan occur is air. The systems and methods provide a means to maintain avolume of a liquid at a predetermined volume, where the volume otherwisewould decrease below the predetermined volume due to evaporation. Thesesystems include a support for performing the reaction; a nanoliterdispensing pipette for dispensing an amount of a liquid onto the surfaceof the support; a temperature controlling device for regulating thetemperature of the support; and means for controlling the amount ofliquid dispensed, wherein the amount of liquid dispensed corresponds tothe amount of liquid that evaporates from the support, wherein thesystem is not sealed.

Analytical Methods

The APL system can be used to perform a number of different reactions,dependent upon the nature of the sample and the analysis to beperformed. The system is typically used to perform analysis onbiological samples, typically biopolymers, including nucleic acids,proteins, peptides and carbohydrates. Methods of analysis of thebiological samples include all known methods of analysis, including, butnot limited to mass spectrometry (all light wavelengths), radiolabeling,mass tags, chemical tags, fluorescence, and chemiluminescence.

In a preferred embodiment, the sample is a purified previously amplifiedportion of genomic DNA or genomic DNA sample. For analysis of DNAsamples, reactions such as nucleic acid amplification (e.g., PCR, ligasechain reaction) and enzymatic reactions, such as primer oligonucleotidebase extension (PROBE), nested PCR or sequencing, may be performed. Inaddition, the apparatus can be used for hybridization (sequencing anddiagnostic) reactions, and endo- and exonuclease mapping of biopolymers.

In certain embodiments, the sample may be immobilized on a solid supportduring all or part of the automated process. For example, enzymaticreactions, including diagnostics, such as a method designated primeroligo base extension (PROBE; see, e.g., published International PCTapplication No. WO 98/20019), nested PCR, sequencing, and otheranalytical and diagnostic procedures that are performed on solidsupports (see, e.g., U.S. Pat. No. 5,605,798). Briefly PROBE uses asingle detection primer followed by an oligonucleotide extension step togive products, which can be readily resolved by MALDI-TOF massspectrometry. The products differ in length by a number of basesspecific for a number of repeat units or for second site mutationswithin the repeated region. The method is exemplified using as a modelsystem the AluVpA polymorphism in intron 5 of the interferon-α receptorgene located on human chromosome 21, and the poly T tract of the spliceacceptor site of intron 8 from the CFTR gene located on human chromosome7. The method is advantageously used for example, for determiningidentity, identifying mutations, familial relationship, HLAcompatibility and other such markers, using PROBE-MS analysis ofmicrosatellite DNA. In a preferred embodiment, the method includes thesteps of a) obtaining a biological sample from two individuals; b)amplifying a region of DNA from each individual that contains two ormore microsatellite DNA repeat sequences; c) ionizing/volatilizing theamplified DNA; d) detecting the presence of the amplified DNA andcomparing the molecular weight of the amplified DNA. Different sizes areindicative of non-identity (i.e. wild-type versus mutation),non-heredity or non-compatibility; similar size fragments indicate thepossibility of identity, of familial relationship, or HLA compatibility.More than one marker may be examined simultaneously, primers withdifferent linker moieties are used for immobilization.

As noted solid supports include, but are not limited to, flat surfaces,microtiter plates, beads, wafers, chips, and silicon support.Compositions and methods for immobilizing nucleic acids to solidsupports, including methods for high density immobilization of nucleicacids are described in U.S. patent application Ser. Nos. 08/746,055 and08/947,801 and published International PCT application No. WO 98/20166.Linkers for immobilizing nucleic acids to solid supports are well known.Linkers may be reversible or irreversible. A target detection site canbe directly linked to a solid support via a reversible or irreversiblebond between an appropriate functionality (L′) on the target nucleicacid molecule (T) and an appropriate functionality (L) on the capturemolecule (FIG. 1B). A reversible linkage can be such that it is cleavedunder the conditions of mass spectrometry (i.e., a photocleavable bondsuch as a charge transfer complex or a labile bond being formed betweenrelatively stable organic radicals).

Photocleavable linkers are linkers that are cleaved upon exposure tolight (see, e.g., Goldmacher et al. (1992) Bioconj. Chem. 3:104-107),thereby releasing the targeted agent upon exposure to light.Photocleavable linkers that are cleaved upon exposure to light are known(see, e.g., Hazum et al. (1981) in Pept., Proc. Eur. Pept. Symp. 16th,Brunfeldt, K (Ed), pp. 105-110, which describes the use of a nitrobenzylgroup as a photocleavable protective group for cysteine; Yen et al.(1989) Makromol. Chem 190:69-82, which describes water solublephotocleavable copolymers, including hydroxypropylmethacrylamidecopolymer, glycine copolymer, fluorescein copolymer and methylrhodaminecopolymer; Goldmacher et al. (1992) Bioconj. Chem. 3:104-107, whichdescribes a cross-linker and reagent that undergoes photolyticdegradation upon exposure to near UV light (350 nm); and Senter et al.(1985) Photochem. Photobiol 42:231-237, which describesnitrobenzyloxycarbonyl chloride cross linking reagents that producephotocleavable linkages), thereby releasing the targeted agent uponexposure to light. In preferred embodiments, the nucleic acid isimmobilized using the photocleavable linker moiety that is cleavedduring mass spectrometry. Exemplary photocleavable linkers are set forthin published International PCT application No. WO 98/20019. Bead linkersfor immobilizing nucleic acids to solid supports are described inallowed U.S. application Ser. No. 08/746,036 and published InternationalPCT application No. WO 98/20166 and WO 98/20020.

Preferred applications include, but are not limited to, sequencing anddiagnostics based on analysis of nucleic acids and polypeptides ordiagnostics by mass spectrometry. Preferred mass spectrometric methodsinclude ionization (I) techniques including, but not limited to, matrixassisted laser desorption (MALDI), continuous or pulsed electrospray(ESI) and related methods (e.g. Ionspray or Thermospray), or massivecluster impact (MCI); the ion sources can be matched with detectionformats including linear or non-linear reflectron time-of-flight (TOF),single or multiple quadruple, single or multiple magnetic sector,Fourier Transform ion cyclotron resonance (FTICR), ion trap, andcombinations thereof (e.g., ion-trap/time-of-flight). For ionization,numerous matrix/wavelength combinations (MALDI) or solvent combinations(ESI) can be employed. DNA sequencing by mass spectrometry is describedin U.S. Pat. Nos. 5,547,835; 5,691,141; and related U.S. applicationSer. Nos. 08/467,208, 08/481,033 and 08/617,010 and in PCT patentapplication No. PCT/US98/26718, filed Dec. 15, 1998, publishedInternational PCT application Nos. WO 94/16101 and WO 97/37041.

DNA sequencing using mass spectrometry is described in U.S. Pat. No.5,547,835. DNA sequencing by mass spectrometry via exonucleasedegradation is described in allowed U.S. application Ser. No.08/744,590, U.S. Pat. No. 5,622,824, published International PCTapplication No. PCT/US94/02938, U.S. Pat. Nos. 5,851,765, and 5,872,003.Processes for direct sequencing during template amplification aredescribed in allowed U.S. patent application Ser. No.08/647,368{circumflex over ( )}C and published International PCTapplication No. WO 97/42348.

DNA diagnostics based on mass spectrometry are described in U.S. Pat.No. 5,605,798 and published International PCT application Nos. WO96/29431 and WO 98/20019. Diagnostics based on mass spectrometricdetection of translated target polypeptides are described in U.S.application Ser. No. 08/922,201 and published International PCTapplication No. WO 99/12040. Mass spectrometric detection ofpolypeptides is described in U.S. patent application Ser. No. 08/922,201and U.S. application Ser. No. 09/146,054.

It is understood that the nature of the sample to be analyzed and theanalysis to be performed, as well as the feasibility of automating areaction process, determine the methods used in the APL, and the methodsare not to be limited to the particular embodiments described herein.Any method and process that requires small volumes and involves one ormore steps in the exemplified embodiment may be adapted and used in anAPL as described herein.

Exemplary Embodiment

One preferred embodiment, which is a dual space system, integratesnucleic acid amplification (via PCR), immobilization of the nucleic acidon a solid support, followed by enzymatic reaction (e.g., PROBE, massarray, sequencing, nested PCR), sample conditioning, addition of anorganic acid matrix for MALDI-TOF analysis and MALDI-TOF analysis on amicrochip. This embodiment is described with respect to the AutomatedProcess Line (APL) system 100 depicted in FIG. 1. As noted above,samples are initially prepared in a contamination-controlled environment102, such as a clean room or laminar flow room, and are moved by asterile transport chamber 104 or taxicab into a non-sterile environment106. In FIG. 1, samples are indicated by rectangular elements withcriss-crossed lines.

In the FIG. 1 embodiment, sample preparation begins in a Liquid HandlingSystem 108, such as the Tecan “Genesis 200/8 Robotic Sample Processor”product. One or more samples 110 of purified genomic DNA are deliveredby a robot 112 to 96-well or 384-well microtiter plates 114 in theLiquid Handling System 108, preferably using a 200 cm instrument widthand an 8-tip arm. These sample processing steps occur in thecontamination-controlled environment 102. Multiple samples may beincluded in the APL system for high-throughput processing. These samplesmay, at times during processing, be held in a sample storage apparatus,such as the “Plate Cube” rack 116 available from Robocon. To the sampleplates 114 are added a PCR reaction mix 118, including PCR primers,where one of the primers is labeled at the 5′ end with functionality,such as biotin, that can be used to immobilize the amplicon to a solidsupport is added to the sample mixture. Where multiple samples are to beprocessed, a wash solution is contained in a reservoir 120 and is usedto clean the pipette tips to prevent cross-contamination between samplesor reagents. Alternatively, the APL system can process multiple samplesusing disposable pipette tips.

The sample plates are manipulated by a robotic system, for example theRobocon robot 112, such as the CRS A 255 Robot, which moves along acentral track 122. The robot 112 operates under control of a clean roomcontrol system computer 124 that includes a central processing unit(CPU) 126, a operator interface 128, and an APL interface 130. The CPUcan comprise any commercially available desktop computer, such as anIBM-compatible personal computer (PC) or the like.

The operator interface 128 includes a visual display and keyboard orother device through which an operator provides commands. The APLinterface 130 is an interface between the computer and the process line,through which the computer 124 controls the robot. The APL interface mayinclude, for example, a robot control program installed in the computer124 and available from Robocon for control of its robot products. Anoptional second computer 131 can assist the first computer 124 inperforming clean room processing.

The robotic arm is equipped with a gripper 132, such as the “DigitalServo Gripper” arm, also available from Robocon, to pick up and drop offthe sample plates 114 as needed, for processing. In a particularembodiment, a microtiter plate is aligned with the gripper so the platereceives pins 134 of the gripper, which more securely couple the platewith the gripper for more secure transport.

FIG. 1 shows a sample plate 140, including the sample and PCR mix, thatis moved to a turntable 142 and oriented such that the robot picks it upand moves it to a bar code reader 144, for example, as is available fromDatalogic, where the bar code is read and recorded for sample tracking.Sample tracking and reorientation may be performed multiple times duringsample processing to assist the robot in sample handling.

The sample plate 140 is reoriented by the robotic arm, using theturntable, and is then placed in a lid parking station 146, such as isavailable as a robotic module in the Robocon robotic system. At the lidparking station, a lid may be parked or retrieved. In the preferredembodiment, the lid is a solid structure, such as a metal lid, with aflexible seal such that placing the lid on the plate seals the contentsof the plate. The sealing eliminates evaporation during subsequentprocessing, such as PCR amplification. Such a sealing apparatus, knownas “MJ Microseal”, is available from MJ Research, Inc. Alternatively,after the sample plate is reoriented, it can be penetrably sealed. Forexample, the sample plate can be covered with a foil wrap that can laterbe penetrated by test probes or the like. A similar penetrable seal canbe provided by a parafilm that is attached to the plate by heat, orother plastic or wax based sealers.

The sealed sample plate is then picked up by the robotic gripper arm andtransported from the laminar flow environment 102 into the taxicabtransport station 104, which provides a sterile environment. First, anentry door opens in the taxicab to permit the robot to place the sampleplate into the taxicab. Once in the taxicab 104, the entry door closesbehind the sample to prevent contamination. Within the taxicab transportstation 104, the sample plate is placed onto and is transported along apneumatically driven stage, and a second door opens to permit the sampleto exit the taxicab into a non-sterile environment. Once outside thesterile taxicab environment, control of sample manipulation istransferred to a second robot 150, also equipped with a gripper 152 andmoving along a center track 153. The sample plate is transported by therobot 150 and is read by a second bar code reader 154 for sampletracking. The second bar code reader 154, as well as a second turntable156, lid park station 158 and sample storage rack 160 are includedoutside the contamination-controlled area 102 for more efficient samplehandling.

The robot 150 operates under control of a PCR Room computer 161 that hasa construction similar to the Clean Room computer 124. Thus, the PCRRoom computer 161 can comprise any commercially available desktopcomputer that can interface with the APL system process line andstations.

After the sample identification code has been read by the bar codereader 152, the sample plate is moved by the system robot 150 to a PCRstation 162, where amplification is carried out. The amplificationreaction can be PCR, ligase chain reaction, etc. In a preferredembodiment, the “MJR Tetrad” thermocycler, available from MJ Research,Inc., is used for PCR amplification. Other PCR thermocycler systems arecommonly known to those of skill in the art and may optionally beintegrated into the system. Methods for DNA amplification are well knownto those of skill in the art. Multiplex PCR can also be carried outusing the system.

After PCR amplification, the plates are removed from the PCR reactionstation 162 by the robot 150. The plates are then moved to the lid parkstation 158, where the lids are removed and unsealed. As noted above,however, a penetrable seal such as a foil wrap or parafilm is analternative to a lid seal, and if removable lids are not used to sealthe plates, then the lid park station is unnecessary and the nextsubstance that must be added to the wells of the plate will be insertedupon piercing of the foil wrap.

Alternatively, using a second liquid handling system 164, preferably aTecan “Genesis 200/8” system, streptavidin-coated paramagnetic beads canbe loaded from a reservoir 166 and mixed with the PCR-amplified DNA inthe sample plate, resulting in immobilization of the amplicon via thefunctionalized (e.g. biotinylated) primer. Beads are used, for example,where the samples are contained in multiwell microtiter plates. Thebeads and PCR products are reacted by shaking, using a shaking apparatus168, such as is available from Robocon, and which is integrated into theAPL system.

The sample plates are then moved to a liquid handling and mixing station170, into which a magnetic lift station 172 has been incorporated, forpost-PCR processing. In a preferred embodiment, the liquid handlingstation is a “Multimek 96” well pipetting station, available fromBeckman. The magnetic lift applies magnets to the sample plate by movingthe magnets up against the bottom of the sample plate, for example, byusing a pneumatic lift, thereby immobilizing the DNA and beads, and thesupernatant is removed. The magnets are then released and liquid isadded to the wells to resuspend the sample. Alternatively, the sampleplate could be moved, for example, by the robot to bring it into contactwith the magnet. The magnet can be a solid surface that interacts withthe entire bottom of the sample plate, or can be designed to morespecifically interact with the individual samples. For example, wherethe sample plate is a 96-well microtiter plate, the magnet can beconfigured as 8 or 12 individual strips so that each strip comes intocontact with the bottom of a single row of wells.

Conventionally, the magnets of the magnet lift station 172 areelongated, strip magnets arranged in rows between sample wells.Alternatively, the magnets can be configured as individual pointmagnets, for example, as disk-shaped magnets arranged into an 8×12 gridof magnets that correspond to the positions of the sample wells in a96-well microtiter plate. This configuration provides an advantage overthe magnetic strip configuration, particularly where small volumes areto be added to the sample. For example, as illustrated in FIG. 2, wheremagnetic strips 202 are used with a multiwell microtiter plate 204, themagnet strips are offset from the center of the sample wells 206, andmagnetic beads 208 concentrate along the sides of the wells.

It is desirable that all beads be concentrated in a location such thatadded liquid makes maximum contact with the samples. If, for example, avolume of sample is removed from the wells and a smaller volume is to besubsequently added, the smaller volume might not be sufficient to washall the beads from the side of the wells, and the sample concentrationcould be affected. FIG. 3 is a plan view of the alternative, preferredembodiment, and shows a portion of the construction that centers adisk-shaped point magnet 302 beneath the center of each sample well in amultiwell microtiter plate. For simplicity of illustration, only a 4×5grid is shown. It should be apparent that by using individual pointmagnets at the bottom of the wells, the beads collect at the bottom ofthe wells and are more easily resuspended, particularly where a smallervolume of liquid is to be added. Multiple rounds of liquid handling areemployed to allow for supernatant removal, denaturation of doublestranded DNA, wash steps and the addition of enzymatic reaction reagents(PROBE).

Returning to FIG. 1, a sample plate 176 is next moved by the roboticsystem to the lid park station 158, and sealed with a lid. Thisoperation is optional and is used, for example, when the sample issubjected to high temperatures in order to prevent evaporation. Thesample plate can otherwise remain open to the environment.

The robot 150 moves the sample plate again to the PCR station 162 andplaces it into a thermocycler of the PCR station. The thermocyclercarries out an enzymatic reaction. The enzymatic reaction can be, forexample, PROBE, nested PCR, primer extension, or sequencing reactions(e.g. Sanger). Details for such enzymatic reactions are commonly knownto those skilled in the art.

After the reaction is complete, the sample plate is removed from thethermocycler of the PCR station 162 and then is returned to the lid parkstation 158 by the robot 150, and the lids are removed and the plateunsealed.

The sample plates are again moved to the liquid handling and mixingstation 170 containing the magnetic lift station 172, which applies themagnets, immobilizing the beads and DNA. The liquid handling and mixingstation then removes the supernatant. The magnets are then released andliquid is added to the wells. Multiple rounds of liquid handling areemployed to allow for washing steps or treatment with ammonium citrate,TRIS, or any other reagent that removes salt ions and replaces them withammonium ions, thereby conditioning the samples prior to massspectrometry. Once conditioned, the primer extension product isdenatured from the immobilized DNA with ammonium hydroxide and releasedinto the supernatant. The ammonium hydroxide reaction is performed forfive minutes at approximately 60° F. The supernatant is removed to aclean sample plate and placed on a shaker 168.

The sample plate is next transported to a sample preparation station 178to prepare it for analysis. In a preferred embodiment, where MALDI-TOFmass spectral analysis is performed, nanoliter or smaller volumes ofsample are dispensed onto pre-made silicon chips to form a microarrayand reacted with matrix. In general, however, the sample may involve anypreparation for use with any analytical method. Nanoliter or smallervolumes are dispensed using piezoelectric pipette, such as the“Nano-Plotter” station, available from GeSiM. Finally, the sample plateis transported to the analytical system, e.g., a mass spectrometer orother spectrometric techniques, such as UV/VIS, IR, fluorescence,chemiluminescence or NMR spectrometry, where sample analysis isperformed.

Several alternatives are possible for preparing a sample for analysisand loading the sample into the analytical system. For example, threeseparate components, including a dispensing apparatus, a sample platformcontaining test samples, and an analytical instrument, can be integratedinto the APL system.

In a preferred embodiment, a nanoliter dispensing apparatus(nano-plotter) 180 of the sample preparation station 178 is used toprepare one or more samples for mass spectral (MS) analysis, preferablyusing MALDI-TOF MS. In preparing a sample for MALDI-TOF analysis, thesample is co-crystallized with a matrix material. The sample is thenloaded into a mass spectrometer 182 on a MS sample platform.Alternatively, the MS platform may be integrated into the massspectrometer, rather than a separately-controlled component. The sampleplatform can be adapted to hold one or more sample analysis vessels,such as microchips.

In another embodiment, the APL system can carry out enzymology directlyon the beads and can directly add matrix to the beads to analyze usingmass spectrometry, where the DNA is ionized directly off the beads. Thiseliminates the need for a nanoliter dispensing station 178 such as theGeSiM “Nano-Plotter”, rather, matrix is added with the liquid handlingsystem 170.

In a preferred embodiment, one or more microchips containing testsamples are prepared by dispensing nanoliter volumes of a sample and anorganic acid matrix onto a chip using a nanoliter dispensing apparatus180, or a nano-plotter, and loading the chips into a mass spectrometer182. Alternative embodiments are possible where (1) one or more testsamples, e.q., on sample chips, are prepared on a sample platform on thenano-plotter and the sample platform is then transferred, e.g., by arobot, into the mass spectrometer; or (2) where one or more sample chipsare prepared on the nano-plotter, transferred to a mass spectrometersample platform station 184 and then inserted into the massspectrometer.

In another embodiment, the APL system can carry out enzymology directlyon a microchip by performing the steps of:

1. Aliquot genomic DNA and transfer to second chamber via taxi;

2. PCR amplify the genomic DNA using previously described steps;

3. Using a liquid handling apparatus (Tecan or GeSim) or pintool add DNAto microchip. The chips are held in a holder that can be manipulated bythe robot;

4. Add PCR reaction mix to chip;

5. Incubate on thermocycler;

6. Wash chip with liquid handling apparatus;

7. Add matrix to chip;

8. Load chip in MALDI; and

9. Ionization/Desorption directly from the chip via MALDI.

Mass Spectrometer Interface

The nano-plotter and mass spectrometer are integrated into the APLsystem 100 and communicate with each other, either directly or via acontrol computer. For example, in one embodiment, commands areautomatically executed from a computer controller to initiate openingand closing of a mass spectrometer entry door (e.g., by using pneumaticsor a motor-driven mechanism) and to initiate loading of a MS sampleplatform into the spectrometer (e.g., by using a robotic arm), where theplatform is either loaded with sample chips directly on a nano-plotter180, or the sample chips are prepared on a nano-plotter 180 and then aretransferred onto a sample platform 184. FIG. 4 shows one implementationof the robotic interface between the nano-plotter and the massspectrometer illustrated in FIG. 1.

In the FIG. 4 embodiment, the samples are automatically transported fromthe sample preparation station 178 to the mass spectrometer 182 by arobotic arm system 410 (not shown in FIG. 1). As described above, thesamples are prepared for the mass spectrometer 182 in the nano-plotter180 and/or the sample platform station 184. When preparation iscomplete, an arm 412 rotates about a pivot base 414 to pick up thesamples from the sample preparation station and then positions them at asample entry station 416 of the mass spectrometer.

Data Analysis

Conventionally, the output of mass spectrometer testing is analyzed byan individual datum-by-datum, so that an individual examines the outputof a sample test and makes a conclusion about the test,sample-by-sample. In the Automated Process Line (APL) described above,the volume of test results is sufficiently large that any individualanalyzing the mass spectrometer output would quickly be unable to keepup with the APL output pace. The APL system of the preferred embodimentperforms computer-automated analysis of mass spectrometer output data todetermine genotype or make another analysis as quickly as the systemproduces test results. The data analysis can continue as long as thesystem is in operation, including on a round-the-clock, 24-hour basis.The APL system performs the test output analysis by automaticallyprocessing the mass spectrum output data of a sample, comparing theoutput data against expected spectrum output values for differentgenotypes, producing a conclusion about the sample genotype based on aconclusion about most likely genotype for the sample, and continuingwith the output data of the next sample.

In the preferred embodiment illustrated in FIG. 1, the data analysis isperformed by a dedicated data analysis computer 188 that receives outputdata from the mass spectrometer 182 and any other pertinent APL stationsor components. The data analysis computer can comprise any commerciallyavailable desktop computer, and can have the same configuration andcomponents as the Clean Room control computer 124 described above. Thus,the data analysis computer 188 includes a CPU having an operatingenvironment in which programs are executed, and also includes anoperator interface with a keyboard and a display.

The process line 100 operates continuously until a stop command isreceived, for a high sample throughout. Therefore, the process lineprovides for emergency situations where an immediate halt is required byproviding halt switches 198 placed around the line. The system also canbe halted by a software halt command that is input by an operator at anyof the control computers 124, 131, 161, 188. The sample preparation,testing, and data analysis otherwise continues unimpeded.

A visual display of the data analysis is depicted in FIG. 5, which showsfrom top to bottom: a graph of two exemplary test spectra against whichoutput data will be compared; a graph of output data picked peaks foranalysis; and a graph of smoothed spectrum data. Those skilled in theart will appreciate that the spectra shown in FIG. 5 correspond tomultiple graphs of mass spectrometer output, wherein the horizontal axis(x-axis) units are in mass per unit charge, also referred to as units ofDaltons, and the vertical axis (y-axis) is in relative intensity ofspectrometer discharge.

The exemplary spectra shown in FIG. 5 relate to male-female genotypes,but those skilled in the art will appreciate that any otherpaired-outcome typing decisions may be the subject of the sampleanalysis.

In FIG. 5, the first test spectra is labeled “Test—Female” andcorresponds to output spectra that might be expected from a female testsubject. The second test spectra is labeled “Test—Male” and correspondsto output spectra that might be expected from a male test subject. Thus,the object of the APL processing will be to determine whether a givensample genotype belongs to a female subject or a male subject. The“Picked Peaks” of FIG. 5 spectra is a display of the mass spectrometeroutput for a particular sample over a predetermined range, to showparticular output peaks. The output peaks shown in the Picked Peaksgraph are selected by the APL system based on input parameters suppliedby the APL operator, as described further below. The bottom spectra ofFIG. 5 is a display of the spectra output after correction processinginitiated by the APL system. It should be understood that theTest—Female and Test—Male graphs of the FIG. 5 display will not changeas the APL system processes the mass spectrometer output data, while thePicked Peaks and Smoothed Spectrum graphs are different for each sampledata, and therefore will generally change with each sample beingprocessed. It also should be understood that the Picked Peaks andSmoothed Spectrum displays can be stopped on any one of the outputgraphs, if the operator wants to view one particular set of graphs. FIG.6 is a flow diagram of the operating steps performed by the APL systemin carrying out the mass spectrometer data analysis, and will be bestunderstood with reference to the FIG. 5 graphs.

The first data analysis step, represented in FIG. 6 by the flow diagrambox numbered 602, is to receive test run input parameters. These areparameters that the APL system will receive from an operator and willapply in processing a run of mass spectrometer output data. That is, theAPL system will use the test run input parameters to evaluate testsamples until the test run parameters are changed by the APL operator.As noted above, a test run might involve producing mass spectrometeroutput and analyzing it on a 24-hours-per-day basis. In the preferredembodiment, the operator provides the test run parameters through agraphical user interface using a display mouse and keyboard of the APLsystem. The test run input parameters received from the operator willinclude the x-axis range in Daltons for the spectrometer output data andx-axis locations of expected peaks that are picked for dataidentification and genotype evaluation. The input parameters will alsoinclude an expected baseline value, defining a noise floor above whichdata should comprise a peak.

In the next processing step, represented by the FIG. 6 flow diagram boxnumbered 604, test data is received for a particular test samplesubmitted to the mass spectrometer of the APL system. A particular testsample may be one well in a 96-well-by-96-well tray, for example. Othertray sizes may be accommodated by the APL.

Those skilled in the art will understand that a mass spectrometerbombards a crystalline-based sample with energy until the samplevaporizes and output products are produced. The output products consistof sample particles that are ionized and projected outwardly todifferent distances from the sample center. The mass spectrometerdetects the distribution of output products having a particular mass perunit charge and assigns a relative intensity to those output products.The mass/charge units are given in Daltons or kiloDaltons (kD). Thus,the mass spectrometer output for a given sample is a sequence of pairednumbers, or x-y values, that specify the detected mass/charge over arange of Daltons (x-axis) and the corresponding relative intensity(y-axis) distribution over that range.

For each set of sample data that is processed, the APL system removesthe residual baseline. This processing is represented by the FIG. 6 flowdiagram box numbered 606, and allows for a rolling baseline that mightotherwise skew the output data. More particularly, with currentprocessing systems, it is possible to misinterpret peaks or spikes, suchas where true data peaks are located in valleys. Conventional programsidentify peaks by detecting data intensity values (see FIG. 5) that aregreater than a baseline value. The data, however, can contain localizedareas in which a peak lies within a valley of a plateau area having anelevated baseline. Peaks that are in such valleys may be missed byconventional programs that do not detect a sufficient difference betweenthe peak height relative to the plateau level. It has been found thatsuch conventional programs may correctly identify peaks up to 80% of thetime, but cannot generally provide greater accuracy due to missed peaks.

To remove the residual baseline and increase accuracy, the APL dataanalysis receives the input parameters that contain the operator'sspecification of where the peaks in the sample experimental resultsshould be located in the mass spectrometer output. The APL system thenexamines the output data where there should be no peaks to find the truebaseline value. The processing represented by the FIG. 6 flow diagrambox numbered 606 therefore includes modeling the baseline of the massspectrometer output with a quadratic equation, based on the test runinputs from the operator. It has been found that a quadratic equation issuperior to using a cubic equation, and also closer than a lower-orderfit, even though very small coefficients are expected for the baselinecurve fit.

For example, the range of interest might be mass spectrometer outputover the range of 4000 to 9000 Daltons. The maximum range and minimumrange would be received as test run inputs. In addition, the expectedpeaks for the sample experimental data over that range of interest wouldbe received as test run inputs. The data concerning expected peaksshould include the peaks that will be produced given the data types forwhich there is testing, and also peaks expected in the output as aresult of primer substances in the sample. Thus, the range of interestshould include output artifacts from primer sources. These primer outputartifacts can serve as landmarks to identify any output shifting. Inaddition to the locations of the expected peaks, the APL system alsoreceives the peak width as in input test run parameter. The APL systemassumes that peaks will be distributed as a gaussian curve, and the peakwidth input parameter indicates the approximate width for each of thosepeaks. In the preferred embodiment, there is one input for all peaks.For example, all peaks may be specified as having a width of 10 Daltons(ten x-axis units).

Next, with the test run parameters that specify the range of interestand the location of peaks, the APL system will identify peak-freeregions in the mass spectrometer output of each sample that correspondto the range of interest, with the data at the peaks removed. Forexample, suppose there are two peaks of interest expected in the outputthat will identify a sample as being one genotype or another. Supposealso that there is an additional peak expected in the output, for primeroutput artifacts. Therefore, a total of three peaks will be expected inthe mass spectrometer output over the range of interest. Then thepeak-free regions would be those regions in the output data along thex-axis over the range of interest, with the data at the three identifiedpeaks deleted. As noted above, the peaks are assumed to be gaussian,with a width value specified in the input parameters. Therefore, thedata for deletion comprises the peaks identified in the test run inputparameters and also an area two peak widths wide on either side of eachidentified peak (peak midline, +/− two peak widths).

It is the mass spectrometer output data with the peaks deleted thatgives the peak-free region, to which the quadratic equation is fitted.Typically, the variable quadratic coefficients would be small, but it ispossible to get contamination from the lower-mass sample particles,which can skew the output. If such contamination is present in theoutput, then the sample output may be skewed so that the peak freeregions will be best modeled by a quadratic equation. It has been foundthat contamination products are best modeled with a quadratic equation,rather than a linear, cubic, or other type of equation.

The technique for determining the coefficients of the quadratic equationfor the best fit to a peak-free baseline is preferably a least squaresfit technique, which will be well-known to those skilled in the art. Inparticular, error minimization using gradient information has been foundsuitable for the least squares fit. Thus, the curve-fit quadraticbaseline equation can be used to produce an expected baseline over themass spectrometer output range of interest. Therefore, as part of thebaseline correction processing represented by the FIG. 6 flow diagrambox numbered 606, at each data point interval along the range ofinterest (e.g., from 4000 to 9000 Daltons), the curve-fit baselineequation is used to calculate a corrected baseline value, which issubtracted from the sample data. The baseline correction occurs over theentire data range, including at the peaks. This produces a new set ofbaseline-corrected sample data values, i.e., a baseline-corrected outputspectrum.

In the next processing step, represented by the FIG. 6 flow diagram boxnumbered 608, a curve is fit to each baseline-corrected peak value inthe mass spectrometer output data. In the preferred embodiment, astandard curve fitting algorithm is used, such as theMarquardt-Levenberg algorithm. This fits a gaussian curve to eachpossible baseline-corrected output peak position. Those skilled in theart will understand that the output of such curve fitting will providecoefficients of a gaussian distribution centered at each peak that willmatch the height of the baseline-corrected output data at that peak, andwill also provide the covariance of the curve-fit height. Thus, the box308 curve fitting will provide, for each peak, equation coefficientsthat give a peak height and a covariance for the equation at that peak.

In the preferred embodiment, the “Picked Peaks” graph in FIG. 5represents all peaks in the mass spectrometer output that have a heightthat exceeds the baseline corrected value generated by the box 606processing, using peaks that are modeled from the box 608 processing.Alternatively, the Picked Peaks graph may represent the peaks in theactual mass spectrometer output that exceed an input threshold value.This latter type of Picked Peaks graph display is the type that istypically provided by mass spectrometer manufacturers, such asBruker-Franzen Analytik GmbH (“Bruker”) of Germany. In the preferredembodiment, the “Smoothed Spectrum” graph of FIG. 5 represents theoutput from the mass spectrometer with default data processing, whichmay include curve smoothing or other data processing provided by themass spectrometer manufacturer. This type of Smoothed Spectrum graph isprovided, for example, as standard output from the Bruker massspectrometer. Alternatively, the Smoothed Spectrum graph may representthe mass spectrometer output with the baseline threshold parametersubtracted, or the actual mass spectrometer output with thequadratic-fit baseline curve subtracted.

In the next processing step, represented by the FIG. 6 flow diagram boxnumbered 610, the APL system determines the probability that the outputdata at each identified peak location is a valid peak. In the preferredembodiment, the peak validation decision is made by comparingprobability density functions (PDF) for the peak-free region and for thefitted peak by constructing gaussian (or normal) probability curves andcomparing them to determine if the data overlaps. If the two curves (thefitted peak and the peak-free region) are substantially free of anyoverlap, then the APL assumes that a true peak has been identified.Otherwise, the fitted “peak” is considered a spurious datum in the noiseof the mass spectrometer output.

More particularly, the PDF of the peak-free region is assumed to be agaussian distribution. The mean height and the standard deviation aredetermined by the mass spectrometer output for the sample in question.The PDF at each identified peak location is assumed to be a gaussiandistribution with the mean height and the standard deviation given bythe curve fitting algorithm described in box 308. The second gaussiancurve will be determined once for each peak. The degree to which the twocurves resemble each other is compared statistically using hypothesistesting that will be well-known to those skilled in the art. The outputof the hypothesis test will be a probability value (from zero to one)that characterizes the peak under consideration. Thus, each peak isassumed to be an independent statistical event.

For example, the comparison uses the baseline curve, which is aquadratic model (peak-free region) having a particular mean height andcorresponding standard deviation. The comparison also uses the gaussianmodel of each peak, having a mean height and standard deviation. If themean values of the two respective curves are different by more than twostandard deviations, then it is assumed there is no overlap for purposesof peak validation. That is, the test peak is a valid peak. If the twocurves are not different in mean by more than two standard deviations,then the identified peak is not a valid peak, but is part of the outputnoise.

After the APL system evaluates the probability for all of the peaks, itwill know the number of peaks that have been identified as valid. Thesystem then determines probabilities for the genotypes underconsideration. The APL system makes a data typing decision based on thepresence or absence of sufficient true or validated peaks to indicateone genotype or the other. This processing is indicated in FIG. 6 by theflow diagram box numbered 612, and is carried out in a probabilisticmanner.

For example, suppose a sample is to be typed as either female or male,and a female is indicated by the presence of an output peak at aposition “A” and the absence of an output peak at a position “B”, whilea male is indicated by a peak at position “A” and also at position “B”.Then the probability of a sample being female is the product of theprobability of a true peak occurring at A and the probability of a peaknot occurring at B. Stated in equation form:

P(female)=P(A)*(1−P(B)).

The probability of a sample being male is then the product of theprobability of a true peak occurring at position A and the probabilityof a true peak occurring at position B, given by the equation:

P(male)=P(A)*P(B).

This analysis is performed automatically by the APL system for each ofthe samples processed by the mass spectrometer. Based on theseprobabilities, the APL system decides whether the mass spectrometeroutput identifies a male or a female. If the probabilities indicate anambiguous outcome, then the mass spectrometer output is consideredinconclusive. In the preferred embodiment, a probability is consideredconclusive if it is at least ten times the probability of thealternative outcome. Thus, if P(female) is greater than ten timesP(male), then the typing decision is for a female. IfP(male)>10*P(female), then the typing decision is for a male.

After the analysis has been performed for a sample subject, the APLsystem checks for additional mass spectrometer output for analysis. Asnoted above, the APL system can support mass spectrometer output at therate of hundreds of output sets per hour. As indicated by the decisionbox 614 in FIG. 6, if more data is present, an affirmative outcome atbox 614, then APL control resumes with receiving the next set of outputdata at the flow diagram box numbered 304. If there is no more massspectrometer output data for analysis or if a system operator indicatesa halt command, a negative outcome at box 614, then the sample run endsand other operation of the APL continues. For example, operation mayreturn to box 602, where more test run input parameters are received andoutput analysis is resumed. Other processing may occur, as desired.

Databases

In cases of high-throughput, the APL stores results of all samples inall runs in a database. The sample run history may be selected forviewing through an APL user interface such as illustrated in FIG. 7. Theuser interface permits review of the database created by one or moresample runs. An example of the user interface to such a database isshown in the screen display of FIG. 8. The database provides a means ofobtaining test output, reaction details, sample details, and assaydetails for each sample under test. For example, shown as outputcollected in the database are the sample plate number, location of thesample well, sample and plate IDs, name, result of genotype matching,and actual spectrum for each sample.

A database analysis system is also integrated into the APL system (seeFIG. 7) and permits a user to (1) create a new run; (2) copy an existingrun; (3) edit or view an existing run; (4) change status or add comment;(5) view the history of a run; and (6) create or edit an assay or test.In the preferred embodiment, the database is supported by a databasemanagement system from Oracle Corporation.

The processes, systems, and products provided herein have been describedabove in terms of a presently preferred embodiment. There are, however,many configurations for automated process lines not specificallydescribed herein but that are apparent from the disclosure herein. Thedisclosure herein is not limited to the particular embodiments describedherein, but rather, is understood to have wide applicability withrespect to automated process lines generally, particularly in the areasof diagnostics and high throughput screening protocols. Allmodifications, variations, or equivalent arrangements andimplementations that are within the scope of the attached claims shouldtherefore be considered within the scope of the invention.

Since modifications will be apparent to those of skill in this art, itis intended that this invention be limited only by the scope of theappended claims.

What is claimed is:
 1. A data analysis system comprising: a computerhaving an operating environment that executes a data analysis programfor processing test results from a process line having a plurality ofprocessing stations, each of which performs a procedure on a biologicalsample contained in a reaction vessel, wherein one of the processingstations comprises a mass spectrometer; a data analysis program forprocessing the test results by receiving test results from one or moreof the processing stations; removing a residual baseline from the testdata from the mass spectrometer for a biological sample; curve fittingeach peak of the biological sample test data to predetermined inputparameters; determining a probability that each peak of the biologicalsample test data is a valid peak; and making a data typing decisionregarding the biological sample in accordance with the determined validpeaks; and a computer interface that receives the test results from theprocess line and provides the test results to the data analysis program,wherein the data analysis program automatically processes the testresults to make a determination regarding the biological sample in thereaction vessel, and continuously performs such processing forbiological samples until a stop instruction is received.
 2. A dataanalysis system of claim 1, wherein the reaction vessel comprises amultiple-well plate.
 3. A data analysis system of claim 1, wherein thedata analysis system displays exemplary test spectra for data types tobe determined by the data analysis system, along with a graph of testdata picked peaks and a graph of smoothed test spectra data for abiological sample.
 4. A data analysis system of claim 1, wherein thedata analysis system receives test run input parameters that determineprocessing until a different set of input parameters are received.
 5. Adata analysis system of claim 4, wherein the data analysis systemdisplays exemplary test spectra for data types to be determined by thedata analysis system, along with a graph of test data picked peaks and agraph of smoothed test spectra data for a biological sample, and theinput parameters specify display parameters.
 6. A data analysis systemof claim 1, wherein the data analysis system removes the residualbaseline from the test data by modeling the baseline of the massspectrometer data with a quadratic equation specified by the inputparameters.
 7. A data analysis system of claim 6, wherein the inputparameters specify a range of data over which the baseline will bemodeled.
 8. A data analysis system as defined in claim 7, wherein thebaseline is modeled over a peak, free region specified by the inputparameters.
 9. A data analysis system as defined in claim 5, wherein thepicked peaks graph represents all peaks in the mass spectrometer outputthat have a height that exceeds the residual baseline corrected data.10. A data analysis system as defined in claim 9, wherein the dataanalysis system validates a peak after comparing a probability densityfunction for the peak free region with a probability density functionfor a fitted peak if the comparison shows that the respectiveprobability density functions overlap by a predetermined amount.
 11. Thesystem of claim 1, wherein the mass spectrometric format is matrixassisted laser desorption time-of-flight analysis (MALDI-TOF).
 12. Adata analysis system of claim 1, wherein the data typing decision is agenotype.
 13. A data analysis system of claim 1, wherein thepredetermined input parameters serve as landmarks to identify outputshifting.
 14. A data analysis system of claim 1, further comprising anoperator interface.